High fidelity of internal strand transfer catalyzed by human immunodeficiency virus reverse transcriptase.

The Journal of Biological Chemistry
Jeffrey J DestefanoA Raja

Abstract

A system to study the fidelity of internal strand transfer events was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. The homology occurred over a 119-base internal region of the donor which coded for the N-terminal portion of the alpha-lac gene. Polymerase chain reaction (PCR) was used to amplify DNA synthesis products. The PCR products were then digested with PvuII and EcoRI and ligated into a vector which had this same region excised. Transformed Escherichia coli were screened for the ability to produce a functional beta-galactosidase protein by blue-white phenotype analysis with white colonies scored as those with errors in alpha-lac. Products synthesized on the donor were used to assess the error rate of human immunodeficiency virus-RT while products transferring to and subsequently extended on the acceptor (transfer products) were used to monitor transfer fidelity. Human immunodeficiency virus-RT made approximately 1 error per 7500 bases copied in the assay. Nucleocapsid protein (NCp), although stimulating strand transfer 3-fold, had no effect on RT fidelity. Transfer products in t...Continue Reading

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Citations

Feb 28, 2001·Virus Research·V R WellsJ J DeStefano
Jun 26, 2012·Virus Research·Redmond P SmythJohnson Mak
Dec 19, 2006·Journal of Biotechnology·Birgit ReiterHelmut Schwab
Jan 24, 2014·Journal of Virology·Timothy E SchlubMiles P Davenport

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